Analysis of the metabolome of Anopheles gambiae mosquito after exposure to Mycobacterium ulcerans

Abstract

Infection with Mycobacterium ulcerans causes Buruli Ulcer, a neglected tropical disease. Mosquito vectors are suspected to participate in the transmission and environmental maintenance of the bacterium. However, mechanisms and consequences of mosquito contamination by M. ulcerans are not well understood. We evaluated the metabolome of the Anopheles gambiae mosquito to profile the metabolic changes associated with bacterial colonization. Contamination of mosquitoes with live M. ulcerans bacilli results in disruptions to lipid metabolic pathways of the mosquito, specifically the utilization of glycerolipid molecules, an affect that was not observed in mosquitoes exposed to dead M. ulcerans. These results are consistent with aberrations of lipid metabolism described in other mycobacterial infections, implying global host-pathogen interactions shared across diverse saprophytic and pathogenic mycobacterial species. This study implicates features of the bacterium, such as the putative M. ulcerans encoded phospholipase enzyme, which promote virulence, survival, and active adaptation in concert with mosquito development, and provides significant groundwork for enhanced studies of the vector-pathogen interactions using metabolomics profiling. Lastly, metabolic and survival data suggest an interaction which is unlikely to contribute to transmission of M. ulcerans by A. gambiae and more likely to contribute to persistence of M. ulcerans in waters cohabitated by both organisms.

Figure 1

(a) PCA plot of untargeted metabolite analysis demonstrates significant clustering of groups based on treatment. Control group in black (upper right); dead M. ulcerans group in red (bottom left); and live M. ulcerans group in green (top left); (b) Bubble plot of total metabolites demonstrates spectral clustering of compounds for all treatment groups. Line separating chart represents P<0.05 cutoff for significance. Oval highlighting significant cluster of features as a function of retention time.

Figure 2. Box and whisker plots of the relative abundance of selected glycerolipid compounds compared by treatment groups.

CTRL: control; Mu-: irradiated M. ulcerans; Mu+: live M. ulcerans.

Figure 3. Phospholipids (PL) are a major constituent of biological membranes.

PL are characterized by a glycerol backbone attached to a phosphodiester group and a polar head group. Phosphatidylcholine (PC) is a functional class of PL molecule characterized by a choline head group. The fatty acid composition of PC can vary, but is generally composed of one saturated fatty acid and one unsaturated fatty acid, attached in the R position (A). Phospholipase hydrolysis of the PC molecule results in a plethora of subunits involved in many downstream signaling and metabolic processes (B). As PC molecules are cleaved by phospholipase A/B/C/D (Pla/b/c/d) (hashed lines), fatty acids are released (C), in addition to other cleavage products such as diacylglycerol (DAG), phosphatidic acid (PA), and choline (D). Fatty acids liberated from PC, DAG, TG, LysoPC can be elongated and/or modified to form ubiquitous signaling molecules. In this study, eicosatrienoic acid and 20-hydroxy eicosatetraenoic acid (hydroxy arachidonic acid) were found to be in lower abundance in groups exposed to M. ulcerans than controls. Molecular diagrams from Kyoto Encyclopedia of Genes and Genomes (www.Kegg.jp/kegg/) [50, 51].

Figure 4. Abundance of eicosanoid compounds in mosquitoes exposed to live (Mu+) and dead (Mu-) M. ulcerans bacteria compared to control mosquitoes.

Eicosanoids are mediators of a wide variety of immune signaling processes, and the down regulation of these compounds is suspected to increase the survival of contaminating pathogens.

Figure 5. Box and whisker plot showing the abundance of riboflavin in treatment groups.

Mosquitoes exposed to live M. ulcerans (Mu+) show a significant accumulation of riboflavin compared to the other groups. CTRL: control, and Mu-: dead M. ulcerans.